Establishment of Stroma-Free Cultures for Human B Lymphopoiesis: Roles of High Cell Density Condition and Mesenchymal Stem Cell-Secreted Factors.

Abstract

Background: Due to lacking of appropriate culture systems, it has been difficult to analyze mechanisms of human B lymphocytes. However, we recently found that human mesenchymal stem cells (MSC) have high ability to support human B lymphopoiesis. Although many investigators have believed that stromal layers are essential for supporting the differentiation of human B lymphocyte progenitors, this hypothesis is not a case. By manipulating our co-culture system, we have successfully established stroma-free suspension cultures, which produce CD10+ B cells from human umbilical cord blood (CB) CD34+ cells.Methods: Our stroma-free culture of CB CD34+ cells was performed in QBSF® medium with 10% FCS in the presence of 10 ng/ml stem cell factor (SCF) and 5 ng/ml Flt3-ligand (FL) with or without 5 ng/ml IL-7.Results:1.Even when co-cultures were separated from MSC with membrane filters, they could produce CD10+ cells. Moreover, addition of MSC supernatant to the cultures permitted CD34+ cells to emerge CD10+ cells in the absence of MSC. Therefore, the cell-cell contact between MSC and B lymphocyte progenitors was not essential.2.For stroma-free human B cell cultures with MSC supernatant, QBSF® was the most suitable and serum components were essential while depending on their lot. Although addition of thymic stromal lymphopoietin or IL-7 increased the production of CD10+ cells, neutralizing antibodies for them showed no effect. Addition of Hemokinin-1 antagonist diminished, albeit to a limited degree, the production of CD10+ cells. Addition of neutralizing antibody for CXCR4 had no effect. Therefore, MSC supernatant contains some supportive factors except for them.3.When cultures without MSC or their supernatant stared at 1x104 cells/ml, they could successfully produce CD10+ cells. The density of the cultured cells was critical. The production of CD10+ cells was not detected when CD34+ cells were seeded at low density (1x103/ml). Moreover, when the cultured cells were diluted and adjusted at 1x104/ml weekly, the emergence of CD10+ cells was not observed while the production of CD33+ myeloid cells was enhanced. Therefore, surrounding hematopoietic cells seemed to be required to support human B lymphopoiesis.4.Our suspension cultures of 1x104 CD34+ cells in the presence of SCF, FL, and IL-7 without any stromal materials generated approximately 0.5–1x106 CD10+ cells at 4 week.CD33+ cells were first expanded within 2 weeks, and then CD10+ cells appeared. When the cultured cells were transplanted into NOD/SCID/γcnull mice, they reconstituted both myeloid and B lymphoid lineages. Therefore, some cultured cells maintain stem cell character.Conclusions: We have established stroma-free suspension cultures, which effectively produce CD10+ B cells from CB CD34+ cells. High cell density condition can in part substitute for stromal layers in supporting human B lymphopoiesis although the addition of MSC supernatant enhances the production of CD10+ cells. Our suspension culture does not use any stromal cells, which produce many positive or negative regulators for human B lymphopoiesis. This simplicity proposes that this culture system is useful in a variety of fields such as the screening direct effects of drugs influencing on human B lymphocyte development and the evaluation of progenitors in patients with B-cell malignancies as well as the cloning of human B lymphocyte-supportive molecules.